PROJECT SUMMARY The inner hair cells reside in a highly polarized ionic environment that is critical for proper transduction of sound waves into neural signals. Potassium-rich endolymph bathes the apical surface of the sensory epithelium, prompting a passive, depolarizing influx of K+ into the hair cells through transduction channels when their stereocilia are deflected, and a repolarizing efflux when K+ flows down its concentration gradient through basally-located K+ channels. The appositional supporting cells are thought to clear K+ and recycle it back into the endolymphatic space through gap-junction coupling, but how supporting cells regulate and remove extracellular K+ remains unclear. This proposal seeks to investigate the novel hypothesis that supporting cells can directly influence the excitability of nearby hair cells by regulating the local K+ concentration in response to extracellular cues. Preliminary data demonstrate that inhibiting the metabotropic purinergic P2Y1 receptor (P2ry1), which appears to be expressed exclusively in supporting cells, results in tonic activation of the inner hair cells. Given the known sensitivity of hair cells to extracellular K+, it is likely that slow K+ accumulation occurs after loss of P2ry1 signaling, leading to a depolarization of hair cells. Using whole-cell electrophysiology, the membrane potential of inner hair cells will be measured after inhibition of P2ry1 and in P2ry1-/- mice to test this hypothesis. Similar experiments will be performed with and without K+ channel blockers in the recording pipette to determine if K+ is mediating this effect. Astrocytes, which maintain extracellular K+ in the brain, increase K+ uptake following purinergic activation and subsequent stimulation of the Na,K-ATPase. Given many similarities between astrocytes and supporting cells, it is possible that similar mechanisms for K+ clearance are employed by these cells. This leads to the hypothesis that P2Y1 inhibition reduces the activity of the Na,K-ATPase in supporting cells, resulting in accumulation of extracellular K+ and tonic activation of hair cells. Preliminary data suggest that the Na,K- ATPase promotes K+ clearance in the supporting cells. Whole-cell electrophysiology in both hair cells and supporting cells will be used to investigate the interaction between Na,K-ATPase and P2ry1. The effect of blocking or removing P2ry1 in ex vivo preps is clear: hair cells become tonically active. To investigate this mechanism in vivo, neural activity of auditory neurons in the inferior colliculus will be imaged using genetically-encoded indicators in P2ry1-/- and wildtype littermate controls. If P2ry1 regulates the excitability of hair cells, increased uncorrelated activity is expected in P2ry1-/- mice. These studies will extend our knowledge about sound transduction, the consequences of purinergic signaling following acoustic trauma, and peripheral tinnitus, which is thought to arise from hyperexcitability in the inner ear.